Among all cardiovascular-related diseases, coronary artery disease remains the leading cause of death in western countries. The incidence and progression of heart disease is different in males and females, indicating that some protective factors are enhanced in one sex. The long term objectives of this project are to identify cellular and genetic mechanisms, regulated by sex chromosomes and gonadal hormones that affect cardiovascular disease, to improve the understanding of endogenous mechanisms of disease, and to identify sex-biased protective factors that may become targets for therapies. Previous research from our labs indicated that the number of X chromosomes causes striking differences in the response of XX and XY mice to myocardial ischemia/reperfusion injury, independent of the gonadal sex of the mice. XX mice show dramatically greater susceptibility to ischemia/reperfusion (I/R) injury, relative to XY mice, and lower post-ischemic heart contractile function. The XX vs. XY difference is attributable to the number of X chromosomes, not the presence/absence of the Y chromosome. Kdm6a is an X chromosome gene that escapes inactivation and is expressed higher in XX than XY cells, and two copies of this gene (as occurs in XX cells) cause greater I/R injury than one copy (as occurs in XY cells). The central hypothesis is that Kdm6a contributes to X chromosome dependent me /R injury via epigenetic regulation. Moreover, because estradiol is a female-enhanced factor that protects from I/R injury, one hypothesis is that estradiol regulates gene targets of KDM6A. Aim 1 will test whether KDM6A is responsible for the XX vs. XY difference in I/R injury by measuring infarct size and heart functional recovery in mice with different copy numbers of Kdm6a (deleting one copy of Kdm6a from XX mice, and adding Kdm6a to XO mice). Aim 2 will discover direct targets of histone demethylase KDM6A, and measure the changes in expression of genes regulated by KDM6A dosage. These studies will define a short list of genes downstream of KDM6A that can be tested to determine if they mediate KDM6A's effects on me /R injury. Aim 3 will compare the effects of two female-specific factors, estradiol secreted from the ovary, and the presence of a second X chromosome, to establish how each factor influences the effect of the other. Genes regulated by KDM6A will be tested to determine if they are also regulated by estradiol. These studies are intended to provide a more sophisticated mechanistic understanding of the differences in I/R injury in females and males, with an eye towards discovering novel protective mechanisms that could become targets for therapy in both sexes.